Determination of Bacterial Endotoxins
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The most famous class of pyrogens is endotoxins or high-molecular complexes localized in the outer membrane of Gram-negative bacteria. They can be both toxic and nontoxic. The problem of determining trace concentrations of endotoxins in pharmaceuticals, biological liquids and water is an important problem of pharmaceutical analysis today. Until now, endotoxins are determined by animal tests, some (inaacurate) physicochemical methods and LAL (Limulus Amebocyte Lisate) test, based on the natural reaction of enzyme cascade by the blood of Limulus Poliphemus species resulting in the formation of a blood clot with the rate of formation proportional to the concentration of endotoxins. Despite its high sensitivity and wide acceptance, LAL test has several drawbacks like the absence of the control over negative and positive outliers, and no data exist on the selectivity of the method, etc. Thus, the development of other sensitive methods of determination of endotoxins is topical.Existing chemical methods of endotoxin determination are based on the determination of specific parts of their molecules like Lipid A, 2-keto-3-desoxioctanic acid, etc. In this study, we estimated the possibility of determination of endotoxins by their polysaccharide part by thermal lensing based on spectrophotometric reactions of polysaccharide. The main aim of this part of our study was to estimate the sensitivity and reproducibility of thermal-lens determination of endotoxinsThe following polysaccharides—plantaglucide, dextran, and geparine—were selected as having the similar chemical properties to polysaccharide residues in lipopolysaccharides. Another test substance is pharmaceutical dosage formulations of Pyrogenal as this medicine is a lipopolysaccharide-series pyrogenic substance with a tabulated concentration in its preparations used for injections.The selected photometric procedure is based on the reaction of polysaccharides with phenol in the presence of concentrated sulfuric acid resulting in the formation of a colored product with the absorption band maximum at 480 nm. The original procedure is characterized by a very high blank signal, thus, at the first stage, it was optimized by diminishing volumes of the test solutions and concentrations (by a factor of five) of phenol and sulfuric acid. This provides a threefold decrease in the blank signal, and a decrease in the analysis time from 60 to 20 min. The limits of detection of model polysaccharides by thermal lensing are 2–5 mg/ml (see Table 1). Table 1. The coefficients of calibration equation А = Xc + Y,coefficients of correlation r and thermal-lens limits of detection of polysaccharides by their reaction with phenol in the presence of sulfuric acid (Р = 0.95)
Remark: *The values in parentheses are increases in the sensitivity compared to conventional spectrophotometric measurements Without any changes, this procedure was used for the determination of total pyrogenic polysaccharides in Pyrogenal dosage formulations. The reproducibility of measurements is 5%, the coefficient of correlation is r = 0,988. As the limit of detection under these conditions corresponds to a low thermal-lens signal against a high blank, we used 10S-criterion for calculating the limit of detection of Pyrogenal. The corresponding limit of detection is 300 ng/ml. Concentrations of Pyrogenal (maximal allowable for injections, minimum used for injections and the limit of detection by the proposed thermal-lens procedure) (P = 0,95; n = 11)
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